Prostaglandin E2 (PGE2) is a potent modulator involved in the pathogenesis of arthritis. PGE2 binds to at least four subtypes of PGE receptor, designated EP1, EP2, EP3, and EP4. Molecular studies have revealed that all subtypes are 7-transmembrane spanning receptors that belong to the G-protein coupled receptor superfamily (Robert et al., Am. Soc. Pharm. Exp. Ther. 46:205–29, 1994). EP1 activation stimulates the release of intracellular calcium via a G protein-mediated mechanism; EP2 and EP4 both activate adenylate cyclase via stimulatory G proteins, but differ in their response to certain ligands; and EP3 inhibits adenylate cyclase via inhibitory G-proteins (Robert et al., supra, Negishi et al., Biochimica Biophys. Acta 1259:109–20, 1995).
Increased levels of interleukin-6 (IL-6), a pleiotropic inflammatory cytokine, have been proposed to contribute to a number of pathological disorders such as rheumatoid arthritis autoimmune diseases and atherosclerosis.
Interleukin-6 (IL-6) is a key cytokine required for plasma cell induction, antibody secretion, B cell growth, activation of acute-phase protein synthesis, T cell activation, hematopoietic stem cell growth and maintaining optical immune function. Its action ensures that the immune system is activated and the activation is sustained during infections and in response to other inflammatory stimuli. It has been well described that IL-6 plays critical role in the development and maintenance of chronic inflammatory disease such as rheumatoid arthritis in mammals. In the recent clinical study, anti-IL-6 receptor antibody improved stiffness, joint pain and swelling in patients with rheumatoid arthritis (Yoshizaki et al., Springer Semin Immunopathol. Vol. 20, 247–259, 1998). This evidence implicating the regulation of IL-6 production provides a promising strategy for the treatment of chronic inflammatory diseases.
Atherosclerosis is a complex disease that is characterized by cholesterol deposition and monocyte infiltration into the subendothelial space, resulting in foam cell formation (Ross R.(1993) Nature 362:801–809). The presence of macrophages and T lymphocytes in the atherosclerotic lesion suggests an important role for the immune system and the inflammatory process in the pathogenesis of atherosclerosis (Libby et al.(1993) Curr. Opin. Lipidol. 4:355–363).
The mRNA transcripts of IL-6 have been detected in human atherosclerotic lesions (Seino et al. Cytokine 1994, 6, 87–91). This observation has been confirmed and extended by immunohistochemical studies that have demonstrated co-localized IL-6 protein expression with macrophages as well as smooth muscle cells in human atherosclerotic plaques (Kishikawa H. et al., Virchows. Arch. A Pathol. Anat. Histopathol. 1992, 423, 433–442). Moreover, IL-6 has been shown to have important effects on the cell types that are components of atherosclerotic lesions. IL-6 can prime THP-1 macrophage cells to produce enhanced amounts of tumor necrosis factor-α in response to lipopolysaccharide (LPS)(Cochran F R et al., Immunopharmacology, 1992,23,97–103), suggesting that IL-6 may play a role in stimulating macrophages to attain their full inflammatory potential. IL-6 has been shown to stimulate the growth of vascular smooth muscle cells in a platelet-derived growth factor-dependent manner (Ikeda U. et al., Am. J. Physiol. 1991,260, H1713–H1717). Recent data demonstrated that the apolipoprotein E-knock out (KO) mouse developed atherosclerosis which is relevant to human pathology (Bourassa P-AK et al., Proc. Natl. Acad Sci USA 1996, 93, 10022–10027; Kauser K. et al., J. Vasc. Res. 1996, 33(suppl 1)48, Abstract). The secretion of IL-6 from isolated aorta from apoE-KO mice showed positive correlation with the lesion area of the same apoE-KO aorta, and immunohistochemical staining revealed that macrophages predominantly produced IL-6 (Sukovich D. A. et al., Arterioscler Thromb Vasc Biol. 1998, 18, 1498–1505). Thus, IL-6 appears to play important role in the development of atherosclerosis.
Additional current data supports the hypothesis that atherosclerosis is an inflammatory disease (Ross, R., et al., Nature, 1993, 362, 801–809; Alexander, R. W., et al., N. Engl. J. Med., 1994, 331, 468–469), and studies examining markers of inflammation demonstrate a relation between increasing inflammation and risk of myocardial infarction (Rider, P. M. et al., N. Engl. J. Med., 1997, 336, 973–979; Liuzzo, G. et al., N. Engl. J. Med., 1994, 331, 417–424). Plaque rupture leading to thrombosis is the key event in infarction and has been shown to be related to increased inflammation within the plaque (van der Wal. A. C., et al., Circulation, 1994, 89, 36–44). Furthermore, reduction in the inflammatory response may be associated with a reduction in the risk of subsequent ischemic events (Ridker, P. M. et al., N. Engl. J. Med., 1997, 336, 973–979), and the beneficial effect of aspirin, a cyclooxygenase inhibitor, in reducing the risk of myocardial infarction has been suggested to be partly attribute to is anti-inflammatory action. This evidence implies that in addition to cytokines and growth factors, prostaglandins also seems to play pivotal role in atherosclerosis.
Prostaglandins are normally produced by the enzyme cyclooxygenase-1 (COX-1), which is constitutively expressed by the vascular endothelium, platelets, kidneys, and elsewhere (Monkada, S. et al., Nature, 1976, 263,663–665). In addition, a cytokine-inducible cyclooxygenase, COX-2, has been detected in several different cell types. The expression of COX-2 is restricted under basal conditions and unregulated during inflammation such as in rheumatoid arthritis (Needeleman, P. et al., J. Rheumatol, 1997, 24(suppl 49), 6–8). In human atherosclerotic lesions, COX-2 was found in macrophages, in some smooth muscle cells and in endothelial cells (Christopher, S. R., et al., Arterioscler Thromb. Vasc. Biol. 1999, 19, 646–655; stemme, V. et al., Eur. J. Vasc. Endovasc, Surg., 2000, 20, 146–152). Since the COX-2 activation produces prostaglandin E2 and I2 which are well recognized as crucial and positive factor in inflammation, such as PGE2 and/or PGI2 might play important role in the atherosclelotic disorders.Several lines of evidence suggest that prostaglandins and IL-6 production from macrophages at inflammatory site or atherosclerotic lesion relates disease development and maintenance. Indeed, it has already reported when monocytes from human peripheral blood were co-stimulated with PGE2 and titanium particles, PGE2 enhanced IL-6 production to a great extent (Blaine, T. A. et al, J. Bone Joint Surgery, 1997, 10, 1519–1528). In the present study, we also show that PGE2 enhances IL-6 production in Concanavalin A (ConA)-treated human peripheral blood mononuclear cells (PBMC). We have surprisingly found that EP4 subtype selective antagonists inhibit the IL-6 production in both PBMC and peripheral whole blood that was co-stimulated with PGE2 and ConA (See DETAILED DESCRIPTION OF THE INVENTION).